This invention relates to a process for lysing membranes of liposomes by using the a reaction of avidin with biotin, and a process for determining an analyte such as an antigen or antibody, a sugar chain or lectin, or nucleic acid or its complementary polynucleotide by applying such a lysis process.
Immunoassay is a measuring method utilizing an antigen-antibody reaction and is widely used, for example, as a method for specifically measuring trace components, e.g. living components such as proteins, hormones, active peptides, autacoid, tumor markers, immunoglobulin, etc., drugs such as digoxin, phenytoin, phenobarbital, etc., in body fluids.
As immunoassays which are now generally used, there are radioimmunoassay (RIA), enzyme immunoassay (EIA), etc. These methods permit quantitative measurement of trace components in samples but involve individual problems. That is, RIA is disadvantageous, for example, in that since radioisotopes should be used therein, RIA requires special facilities and troublesome disposal of wastes. EIA is disadvantageous, for example, in that it requires a relatively long measuring time and is difficult to apply to an autoanalyzer.
Therefore, as an immunoassay involving none of these problems, there has recently been proposed and noted an immunoassay using liposomes (hereinafter referred to as "liposome immunoassay"). A typical example of this method is disclosed in Japanese Patent Unexamined Publication No. 56-132564 (U.S. Pat. No. 4,342,826). This method comprises mixing liposomes, surfaces of which are fixed with an analyte to be measured and which contain a marker (e.g. enzyme) therein, a sample and an antibody to an analyte to carry out the antigen-antibody reaction and adding complement thereto. Thus, complement activated by an antigen-antibody complex formed on the surfaces of liposomes, lyses liposome membranes to liberate the marker encapsulated in the liposomes, thereby measuring the amount of analyte in the sample from the amount of the marker liberated. This liposome immunoassay using complement (hereinafter referred to as "complement immunoassay") does not include the above-mentioned problems of RIA and EIA and can accomplish a series of reactions in a uniform reaction system, so that this method is noticed for carrying out the measurement simply and in a short time. But, according to the complement immunoassay, there is a problem in stability of reagents for measurement since complement per se is an unstable substance. Further, when a sample contains a substance which reacts with a measuring reagent nonspecifically to activate complement and ruptures a part of liposomes to liberate the marker, there arises a problem of causing measuring errors.
In order to solve such problems, there has been developed liposome immunoassay using a substance having a liposome membrane lysis activity other than complement (hereinafter referred to as "membrane lysis agent"). Such a method uses cytolysin typified by melitin which is a major component of bee toxin and reported by Litchfield et al, "High Sensitive Immunoassay Based on Use of Liposomes without Complement" Clin Chem 30, 1441-1445 (1984). This method comprises preparing a complex of a melitin molecule and an analyte, reacting the melitin-analyte complex with a free analyte competitively with an antibody to the analyte by applying a property that when the melitin-analyte complex is bound to the antibody, the membrane lysis activity of melitin is lost, and measuring the retaining liposome membrane lysis activity of melitin so as to determine the amount of the analyte. But this method also has problems in that melitin is difficult to obtain, sufficient liposome membrane lysis activity cannot be obtained unless melitin in a high concentration is used, etc. Thus, this method is not a practically usable method.